Tissue biopsies are frequently obtained for diagnostic and therapeutic purposes. Typically, a tissue biopsy sample consists of a 5 to 10 micron slice of tissue that is placed on a glass microscope slide using techniques well known in the field of pathology. The tissue sample will typically consist of a variety of different types of cells. Often a pathologist will desire to remove only a small portion of the tissue for further analysis. Before the advent of laser microdissection, pathologists would have to resort to various time-consuming and imprecise microdissection techniques to obtain a sample of the desired region of a biopsy. Laser microdissection provides a simple method for the procurement of selected human cells from a heterogeneous population contained on a typical histopathology biopsy slide. The laser microdissection technique is generally described in the published article: Laser Capture Microdissection, Science, Volume 274, Number 5289, Issue 8, pp 998-1001, published in 1996, incorporated herein by reference, and in the following U.S. Pat. Nos. 5,859,699, 5,985,085, 6,184,973, 6,157,446, 6,215,550, 6,459,779, 6,495,195, 6,512,576 and 6,528,248 all herein incorporated by reference in their entirety.
Laser microdissection systems generally comprise an inverted microscope fitted with an infrared laser. Tissue samples are mounted on a standard glass slide and a transparent thermoplastic transfer film is placed over the section. This film is often manufactured containing organic dyes that are chosen to selectively absorb in the near infrared region of the spectrum overlapping the emission region of common AlGaAs laser diodes. When the film is exposed to the focused laser beam the exposed region is heated by the laser and melts, adhering to the tissue in the region that was exposed.
The laser melts the film in precise locations which serves to bind the film to a targeted cell or cells. Individual cells or clusters of cells can be targeted by the laser depending on the diameter of light emitted from the laser. Heat generated by the laser is dissipated by the film, thus limiting the damage done to the targeted cells and the components therein. After the targeted cells are bound to the transfer film, isolation and separation of the targeted cells from the sample occur when the film with the adhered targeted cells are removed from the sample. The targeted cells are then extracted for further analysis. The transfer film can be mounted on a transparent cap that fits on a microcentrifuge tube to facilitate extraction.
The following invention is a new method for laser microdissection that solves a number of problems of conventional laser microdissection and provides the ability to capture moisture-containing samples including live cells from cell cultures. As a practical example, this method allows for colonies of cells grown in slightly modified microtiter plates to be culled using a modified laser microdissection process. The invention also provides a method of capturing cells with improved visualization and a means of minimizing non-specific transfer of unwanted cells without the need for non-contact films or methods designed to space the transfer film from the sample.